Molecules that tightly and specifically bind to the biological targets are of great importance in a wide variety of biochemical, biological and biomedical applications. One of the major challenges in developing a targeting ligand is to improve its target-binding strength. Although such properties can be improved through extensive affinity maturation and engineering, the process is a slow, tedious, and often limited process. An important parameter for satisfactory in vivo targeting is the valency of the targeting ligand, which is defined as the number of antigen-binding sites. Evidence suggests that multivalency is useful for favorable biodistribution and pharmacokinetics in therapeutic and imaging applications. For example, it has been demonstrated that monovalent binding is often not enough to achieve desired cancer targeting, and most monovalent targeting ligands, even those with very high binding affinities, tend to have fast dissociation rates and provide only modest retention time on the target antigen in an in vivo non-equilibrium environment1. One of the most effective approaches used in nature to achieve strong binding between an antigen and its antibody is through multivalent interactions. Significantly, multivalency is an important intrinsic characteristic of natural antibodies in mammals. Although antibodies in camelidae and shark contain only heavy chains, they could acquire divalency through homodimerization. Some of the five naturally occurring antibody classes may form additionally dimer (IgA) or pentamer (IgM) complexes with tetravalency or decavalency, respectively. Naturally occurring IgM antibodies, for example, bind to antigens very tightly and efficiently, although the antigen-binding affinity of its monomeric form is relatively weak. This functional affinity or avidity of multiple antibody-antigen interactions when more than one interaction takes place between two molecules can be orders of magnitude higher than the intrinsic affinity of a single antibody-antigen interaction2. Multivalent targeting ligands have several major advantages over monovalent ligands in interaction with many cancer biomarkers that are present on cell surface. First, the target-binding strength of the multivalent ligands could be significantly improved. In principle, the resulting binding strength (avidity) can reach the product of original binding constants (affinities)3. Second, the multimerization process is often simultaneously accompanied with an increase of the molecular weight by several folds. This is particularly phenomenal for target-binding peptides or small domain-based antibody mimics. For example, the pentamerization of a 20 kDa targeting ligand results in a complex with 100 kDa, which is presumably less efficiently taken up and cleared by kidney4.
During the past 15 years, several techniques in multivalency engineering of antibodies have been developed, including domain-swapping, linear fusion, chemical linking, self-association, and heterodimerization1. Compared to most of these methods that are limited to targeting ligands based on natural antibodies or their fragments, the self-association is a very general approach. Some multimerization domains have been successfully applied to generate multivalent antibody fragments, including TNF-alpha for the formation of homotrimers, the amphipathic helix of GCN4, the multimerization peptide of p53 and the core domain of streptavidin for the formation of tetramers, and the coiled-coil assembly domain of cartilage oligomeric matrix protein (COMP) and the B-subunit of bacterial verotoxin for the formation of pentamers5-12. Despite numerous advantages of using multivalent targeting ligands, successful and efficient conversion of a monovalent ligand into its multivalent form is challenging and requires a combination of unique features on the target-binding and the multimerization moieties. Due to the tendency of aggregation and steric hindrance, few multimerization domains are suitable for efficient multimerization. First, the scaffold should be small and soluble enough with a high expression level in bacteria. Second, the self-assembly of the monomeric domain into a multimeric structure with desired valency should be very efficient with high association constants and low aggregation tendency. The resulting complex should have a well defined parallel multimeric structure with high stability that allows for the introduction of a target-binding moiety and hinge region to achieve desired multivalency without disrupting the overall structure. This is particularly challenging when the complex is significantly diluted in the bloodstream under in vivo conditions. To circumvent these problems, new multimerization domains need to be identified for the generation of targeting ligands with higher avidity.
The present invention overcomes previous shortcomings in the art by providing heptameric targeting ligands that bind to cell surface molecules with high affinity and specificity, for use as therapeutic agents, imaging and/or diagnostic agents and/or as agents to deliver therapeutic agents to target cells.